Mutant luciferases

ABSTRACT

The invention provides active, non-naturally occurring mutants of beetle luciferases and DNAs which encode such mutants. A mutant luciferase of the invention differs from the corresponding wild-type luciferase by producing bioluminescence with a wavelength of peak intensity that differs by at least 1 nm from the wavelength of peak intensity of the bioluminescence produced by the wild-type enzyme. The mutant luciferases and DNAs of the invention are employed in various biosensing applications.

TECHNICAL FIELD

[0001] This invention generally relates to luciferase enzymes thatproduce luminescence, like that from fireflies. More particularly, theinvention concerns mutant luciferases of beetles. The mutant luciferasesof the invention are made by genetic engineering, do not occur innature, and, in each case, include modifications which cause a change incolor in the luminescence that is produced. The luciferases of theinvention can be used, like their naturally occurring counterparts, toprovide luminescent signals in tests or assays for various substances orphenomena.

BACKGROUND OF THE INVENTION

[0002] The use of reporter molecules or labels to qualitatively orquantitatively monitor molecular events is well established. They arefound in assays for medical diagnosis, for the detection of toxins andother substances in industrial environments, and for basic and appliedresearch in biology, biomedicine, and biochemistry. Such assays includeimmunoassays, nucleic acid probe hybridization assays, and assays inwhich a reporter enzyme or other protein is produced by expression undercontrol of a particular promoter. Reporter molecules, or labels in suchassay systems, have included radioactive isotopes, fluorescent agents,enzymes and chemiluminescent agents.

[0003] Included in the assay system employing chemiluminescence tomonitor or measure events of interest are assays which measure theactivity of a bioluminescent enzyme, luciferase.

[0004] Light-emitting systems have been known and isolated from manyluminescent organisms including bacteria, protozoa, coelenterates,molluscs, fish, millipedes, flies, fungi, worms, crustaceans, andbeetles, particularly click beetles of genus Pyrophorus and thefireflies of the genera Photinus, Photuris, and Luciola. In many ofthese organisms, enzymes catalyze monooxygenations and utilize theresulting free energy to excite a molecule to a high energy state.Visible light is emitted when the excited molecule spontaneously returnsto the ground state. This emitted light is called “bioluminescence.”Hereinafter it may also be referred to simply as “luminescence.”

[0005] The limited occurrence of natural bioluminescence is an advantageof using luciferase enzymes as reporter groups to monitor molecularevents. Because natural bioluminescence is so rare, it is unlikely thatlight production from other biological processes will obscure theactivity of a luciferase introduced into a biological system. Therefore,even in a complex environment, light detection will provide a clearindication of luciferase activity.

[0006] Luciferases possess additional features which render themparticularly useful as reporter molecules for biosensing (using areporter system to reveal properties of a biological system). Signaltransduction in biosensors (sensors which comprise a bilogicalcomponent) generally involves a two step process: signal generationthrough a biological component, and signal transduction andamplification through an electrical component. Signal generation istypically achieved through binding or catalysis. Conversion of thesebiochemical events into an electrical signal is typically based onelectrochemical or caloric detection methods, which are limited by thefree energy change of the biochemical reactions. For most reactions thisis less than the energy of hydrolysis for two molecules of ATP, or about70 kJ/mole. However, the luminescence elicited by luciferases carries amuch higher energy content. Photons emitted from the reaction catalyzedby firefly luciferase (560 nm) have 214 Kj/einstein. Furthermore, thereaction catalyzed by luciferase is one of the most efficientbioluminescent reactions known, having a quantum yield of nearly 0.9.This enzyme is therefore an extremely efficient transducer of chemicalenergy.

[0007] Since the earliest studies, beetle luciferases, particularly thatfrom the common North American firefly species Photinus pyralis, haveserved as paradigms for understanding of bioluminescence. Thefundamental knowledge and applications of luciferase have been based ona single enzyme, called “firefly luciferase,” derived from Photinuspyralis. However, there are roughly 1800 species of luminous beetlesworldwide. Thus, the luciferase of Photinus pyralis is a single exampleof a large and diverse group of beetle luciferases. It is known that allbeetle luciferases catalyze a reaction of the same substrate, apolyheterocyclic organic acid,D-(−)-2-(6′-hydroxy-2′-benzothiazolyl)-Δ²-thiazoline-4-carboxylic acid(hereinafter referred to as “luciferin”, unless otherwise indicated),which is converted to a high energy molecule. It is likely that thecatalyzed reaction entails the same mechanism in each case.

[0008] The general scheme involved in the mechanism of beetlebioluminescence appears to be one by which the production of light takesplace after the oxidative decarboxylation of the luciferin, throughinteraction of the oxidized luciferin with the enzyme. The color of thelight apparently is determined by the spatial organization of theenzyme's amino acids which interact with the oxidized luciferin.

[0009] The luciferase-catalyzed reaction which yields bioluminescence(hereinafter referred to simply as “the luciferase-luciferin reaction”)has been described as a two-step process involving luciferin, adenosinetriphosphate (ATP), and molecular oxygen. In the initial reaction, theluciferin and ATP react to form luciferyl adenylate with the eliminationof inorganic pyrophosphate, as indicated in the following reaction:

E+LH₂+ATP

E·LH−AMP+PP_(i)

[0010] where E is the luciferase, LH₂ is luciferin, and PPi ispyrophosphate. The luciferyl adenylate, LH₂−AMP, remains tightly boundto the catalytic site of luciferase. When this form of the enzyme isexposed to molecular oxygen, the enzyme-bound luciferyl adenylate isoxidized to yield oxyluciferin (L=0) in an electronically excited state.The excited oxidized luciferin emits light on returning to the groundstate as indicated in the following reaction:

[0011] One quantum of light is emitted for each molecule of luciferinoxidized. The electronically excited state of the oxidized luciferin isa characteristic state of the luciferase-luciferin reaction of a beetleluciferase; the color (and, therefore, the energy) of the light emittedupon return of the oxidized luciferin to the ground state is determinedby the enzyme, as evidenced by the fact that various species of beetleshaving the same luciferin emit differently colored light.

[0012] Luciferases have been isolated directly from various sources. ThecDNAs encoding luciferases of various beetle species have been reported.(See de Wet et al., Molec. Cell. Biol 7, 725-737 (1987); Masuda et al.,Gene 77, 265-270 (1989); Wood et al., Science 244, 700-702 (1989)). Withthe cDNA encoding a beetle luciferase in hand, it is entirelystraightforward for the skilled to prepare large amounts of theluciferase by isolation from bacteria (e.g., E. coli), yeast, mammaliancells in culture, or the like, which have been transformed to expressthe cDNA. Alternatively, the cDNA, under control of an appropriatepromoter and other signals for controlling expression, can be used insuch a cell to provide luciferase, and ultimately bioluminescencecatalyzed thereby, as a signal to indicate activity of the promoter. Theactivity of the promoter may, in turn, reflect another factor that issought to be monitored, such as the concentration of a substance thatinduces or represses the activity of the promoter. Various cell-freesystems, that have recently become available to make proteins fromnucleic acids encoding them, can also be used to make beetleluciferases.

[0013] Further, the availability of cDNAS encoding beetle luciferasesand the ability to rapidly screen for cDNAS that encode enzymes whichcatalyze the luciferase-luciferin reaction (see de Wet et al., supra andWood et al., supra) also allow the skilled to prepare, and obtain inlarge amounts, other luciferases that retain activity in catalyzingproduction of bioluminescence through the luciferase-luciferin reaction.These other luciferases can also be prepared, and the cDNAs that encodethem can also be used, as indicated in the previous paragraph. In thepresent disclosure, the term “beetle luciferase” or “luciferase” meansan enzyme that is capable of catalyzing the oxidation of luciferin toyield bioluminescence, as outlined above.

[0014] The ready availability of cDNAS encoding beetle luciferases makespossible the use of the luciferases as reporters in assays employed tosignal, monitor or measure genetic events associated with transcriptionand translation, by coupling expression of such a cDNA, and consequentlyproduction of the enzyme, to such genetic events.

[0015] Firefly luciferase has been widely used to detect promoteractivity in eucaryotes. Though this enzyme has also been used inprocaryotes, the utility of firefly luciferase as genetic reporter inbacteria is not commonly recognized. As genetic reporters, beetleluciferases are particularly useful since they are monomeric products ofa single gene. In addition, no post-translational modifications arerequired for enzymatic activity, and the enzyme contains no prostheticgroups, bound cofactors, or disulfide bonds. Luminescence from E.colicontaining the gene for firefly luciferase can be triggered by addingthe substrate luciferin to the growth medium. Luciferin readilypenetrates biological membranes and cannot be used as a carbon ornitrogen source by E.coli. The other substrates required for thebioluminescent reaction, oxygen and ATP, are available within livingcells. However, measurable variations in luminescence color fromluciferases would be needed for systems which utilize two or moredifferent luciferases as reporters (signal geneators).

[0016] Clones of different beetle luciferases, particularly of a singlegenus or species, can be utilized together in bioluminescent reportersystems. Expression in exogenous hosts should differ little betweenthese luciferases because of their close sequence similarity. Thus, inparticular, the click beetle luciferases may provide a multiple reportersystem that can allow the activity of two or more different promoters tobe monitored within a single host, or for different populations of cellsto be observed simultaneously. The ability to distinguish each of theluciferases in a mixture, however, is limited by the width of theiremissions spectra.

[0017] One of the most spectacular examples of luminescence colorvariation occurs in Pyrophorus plagiophthalamus, a large click beetleindigenous to the Caribbean. This beetle has two sets of light organs, apair on the dorsal surface of the prothorax, and a single organ in aventral cleft of the abdomen. Four different luciferase clones have beenisolated from the ventral organ. The luciferin-luciferase reactionscatalyzed by these enzymes produces light that ranges from green toorange.

[0018] Spectral data from the luciferase-luciferin reaction catalyzed bythese four luciferases show four overlapping peaks of nearly evenspacing, emitting green (peak intensity: 546 nanometers), yellow-green(peak intensity: 560 nanometers), yellow (peak intensity: 578nanometers) and orange (peak intensity: 593 nanometers) light. Therespective proteins are named LucPplGR, LucPplYG, LucPplYE and LucPplOR.Though the wavelengths of peak intensity of the light emitted by theseluciferases range over nearly 50 nm, there is still considerable overlapamong the spectra, even those with peaks at 546 and 593 nm. Increasingthe difference in wavelength of peak intensity would thus be useful toobtain greater measurement precision in systems using two or moreluciferases.

[0019] The amino acid sequences of the four luciferases from the ventralorgan are highly similar. Comparisons of the sequences show them to be95 to 99% identical.

[0020] It would be desirable to enhance the utility of beetleluciferases for use in systems using multiple reporters to effectmutations in luciferase-encoding cDNAs to produce mutant luciferaseswhich, in the luciferase-luciferin reaction, produce light withdifferences between wavelengths of peak intensity that are greater thanthose available using currently available luciferases.

[0021] Beetle luciferases are particularly suited for producing thesemutant luciferases since color variation is a direct result of changesin the amino acid sequence.

[0022] Mutant luciferases of fireflies of genus Luciola are known in theart. Kajiyama et al., U.S. Pat. Nos. 5,219,737 and 5,229,285.

[0023] In using luciferase expression in eukaryotic cells forbiosensing, it would be desirable to reduce transport of the luciferaseto peroxisomes. Sommer et al., Mol. Biol. Cell 3, 749-759 (1992), havedescribed mutations in the three carboxy-terminal amino acids of P.pyralis luciferase that significantly reduce peroxisome-targeting of theenzyme.

[0024] The sequences of cDNAs enoding various beetle luciferases, andthe amino acid sequences deduced from the cDNA sequences, are known, asindicated in Table I. Luciferase Reference LucPplGR K. Wood, Ph.D.Dissertation, University of California, San Diego (1989), see also SEQID NO:1; Wood et al., Science 244, 700-702 (1989) LucPplYG K. Wood,Ph.D. Dissertation, University of California, San Diego (1989); Wood etal., Science 244, 700-702 (1989) LucPplYE K. Wood, Ph.D. Dissertation,University of California, San Diego (1989); Wood et al., Science 244,700-702 (1989) LucPplOR K. Wood, Ph.D. Dissertation, University ofCalifornia, San Diego (1989); Wood et al., Science 244, 700-702 (1989)Photinus pyralis de Wet et al., Mol. Cell. Biol. 7, 725-737 (1987); K.Wood, Ph.D. Dissertation, University of California, San Diego (1989);Wood et al., Science 244, 700- 702 (1989) Luciola cruciata Kajiyama etal., U.S. Pat. No. 5,229,285; Masuda et al., U.S. Pat. No. 4,968,613Luciola lateralis Kajiyama et al., U.S. Pat. No. 5,229,285 Luciolamingrelica Devine et al., Biochim. et Biophys. Acta 1173, 121-132 (1993)

[0025] The cDNA and amino acid sequences of LucPplGR, the green-emittingluciferase of the elaterid beetle Pyrophorus plagiophthalamus, are shownin SEQ ID NO:1 (See FIG. 1).

SUMMARY OF THE INVENTION

[0026] The present invention provides mutant luciferases of beetles andDNAs which encode the mutant luciferases. Preferably, the mutantluciferases produce a light of different color from that of thecorresponding wild-type luciferase and preferably this difference incolor is such that the wavelength of peak intensity of the luminescenceof the mutant differs by at least 1 nm from that of the wild-typeenzyme.

[0027] The mutant luciferases of the invention differ from thecorresponding wild-type enzymes by one or more, but typically fewer thanthree, amino acid substitutions. The luciferases of the invention mayalso entail changes in one or more of the three carboxy-terminal aminoacids to reduce peroxisome targeting.

[0028] In one surprising aspect of the invention, it has been discoveredthat combining in a single mutant two amino acid substitions, each ofwhich, by itself, occasions a change in color (shift in wavelength ofpeak intensity) of bioluminescence, causes the mutant to have a shift inwavelength of peak intensity that is greater than either shift caused bythe single amino acid substitutions.

[0029] cDNAs encoding the mutant luciferases of the invention may beobtained straightforwardly by any standard, site-directed mutagenesisprocedure carried out with a cDNA encoding the corresponding wild-typeenzyme or another mutant. The mutant luciferases of the invention can bemade by standard procedures for expressing the cDNAs which encode themin prokaryotic or eukaryotic cells.

[0030] A fuller appreciation of the invention will be gained uponexamination of the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0031] In the following description and examples, process steps arecarried out and concentrations are measured at room temperature (about20° C. to 25° C.) and atmospheric pressure unless otherwise specified.

[0032] All amino acids referred to in the specification, except thenon-enantiomorphic glycine, are L-amino acids unless specifiedotherwise. An amino acid may be referred to using the one-letter orthree-letter designation, as indicated in the following Table II. TABLEII Designations for Amino Acids Three-Letter One-Letter Amino AcidDesignation Designation L-alanine Ala A L-arginine Arg R L-asparagineAsn N L-aspartic acid Asp D L-cysteine Cys C L-glutamic acid Glu EL-glutamine Gln Q glycine Gly G L-histidine His H L-isoleucine Ile IL-leucine Leu L L-lysine Lys K L-methionine Met M L-phenylalanine Phe FL-proline Pro P L-serine Ser S L-threonine Thr T L-tryptophan Trp WL-tyrosine Tyr Y L-valine Val V

[0033] “X” means any one of the twenty amino acids listed in Table II.

[0034] Peptide or polypeptide sequences are written and numbered fromthe initiating methionine, which is numbered “1,” to thecarboxy-terminal amino acid.

[0035] A substitution at a position in a polypeptide is indicated with[designation for original amino acid]_([position number])[designationfor replacing amino acid]. For example, substitution of an alanine atposition 100 in a polypeptide with a glutamic acid would be indicated byAla₁₀₀Glu or A₁₀₀E. Typically, the substitution will be preceded by adesignation for the polypeptide in which the substitution occurs. Forexample, if the substitution A₁₀₀E occurs in an hypothetical proteindesignated “Luck,” the substitution would be indicated as Luck-Ala₁₀₀Gluor Luck-A₁₀₀E. If there is more than one substitution in a polypeptide,the indications of the substitutions are separated by slashes. Forexample, if the hypothetical protein “Luck” has a substitution ofglutamic acid for alanine at position 100 and a substitution ofasparagine for lysine at position 150, the polypeptide with thesubstitutions would be indicated as Luck-Ala₁₀₀Glu/Lys₁₅₀Asn orLuck-A₁₀₀E/K₁₅₀N. To indicate different substitutions at a position in apolypeptide, the designations for the substituting amino acids areseparated by commas. For example, if the hypothetical “Luck” hassubstitutions of glutamic acid, glycine or lysine for alanine atposition 100, the designation would be Luck-Ala₁₀₀/Glu, Gly, Lys orLuck-A₁₀₀/E, G, K.

[0036] The standard, one-letter codes “A,” “C,” “G,” and “T” are usedherein for the nucleotides adenylate, cytidylate, guanylate, andthymidylate, respectively. The skilled will understand that, in DNAs,the nucleotides are 2′-deoxyribonucleotide-5′-phosphates (or, at the5′-end, triphosphates) while, in RNAs, the nucleotides areribonucleotide-5′-phosphates (or, at the 5′-end, triphosphates) anduridylate (U) occurs in place of T. “N” means any one of the fournucleotides.

[0037] Oligonucleotide or polynucleotide sequences are written from the5′-end to the 3′-end.

[0038] The term “mutant luciferase” is used herein to refer to aluciferase which is not naturally occurring and has an amino acidsequence that differs from those of naturally occurring luciferases.

[0039] In one of its aspects, the present invention is a mutant beetleluciferase which produces bioluminescence (i.e., catalyzes the oxidationof luciferin to produce bioluminescence) which has a shift in wavelengthof peak intensity of at least 1 nm from the wavelength of peak intensityof the bioluminescence produced by the corresponding wild-typeluciferase and has an amino acid sequence that differs from that of thecorresponding wild-type luciferase by a substitution at one position orsubstitutions at two positions; provided that, if there is asubstitution at one position, the position corresponds to a position inthe amino acid sequence of LucPplGR selected from the group consistingof position 214, 215, 223, 224, 232, 236, 237, 238, 242, 244, 245, 247,248, 282, 283 and 348; provided further that, if there are substitutionsat two positions, at least one of the positions corresponds to aposition in the amino acid sequence of LucPplGR selected from the groupconsisting of position 214, 215, 223, 224, 232, 236, 237, 238, 242, 244,245, 247, 248, 282, 283 and 348; and provided that the mutant optionallyhas a peroxisome-targeting-avoiding sequence at its carboxy-terminus.

[0040] Exemplary mutant luciferases of the invention are those of thegroup consisting of LucPplGR-R₂₁₅H, -R₂₁₅G, -R₂₁₅T, -R₂₁₅M, -R₂₁₅P,-R₂₁₅A, -R₂₁₅L, -R₂₂₃L, -R₂₂₃Q, -R₂₂₃M, -R₂₂₃H, -V₂₂₄I, -V₂₂₄S, -V₂₂₄F,-V₂₂₄Y, -V₂₂₄L, -V₂₂₄H, -V₂₂₄G, -V₂₃₂E, -V₂₃₆H, -V₂₃₆W, -Y₂₃₇S, -Y₂₃₇C,-L₂₃₈R, -L₂₃₈M, -L₂₃₈Q, -L₂₃₈S, -L₂₃₈D, -H₂₄₂A, -F₂₄₄L, -G₂₄₅S, -G₂₄₅E,-S₂₄₇H, -S₂₄₇T, -S₂₄₇Y, -S₂₄₇F, -I₂₄₈R, -I₂₄₈V, -I₂₄₈F, -I₂₄₈T, -I₂₄₈S,-I₂₄₈N, -H₃₄₈N, -H₃₄₈Q, -H₃₄₈E, -H₃₄₈C, -S₂₄₇F/F₂₄₆L, -S₂₄₇F/I₂₄₈C,-S₂₄₇F/I₂₄₈T, -V₂₂₄F/R₂₁₅G, -V₂₂₄F/R₂₁₅T, -V₂₂₄F/R₂₁₅V, -V₂₂₄F/R₂₁₅P,-V₂₂₄F/P₂₂₂S, -V₂₂₄F/Q₂₂₇E, -V₂₂₄F/L₂₃₈V, -V₂₂₄F/L₂₃₈T, -V₂₂₄F/S₂₄₇G,-V₂₂₄F/S₂₄₇H, -V₂₂₄F/S₂₄₇T, and -V₂₂₄F/S₂₄₇F.

[0041] The following Table III shows spectral properties of these andother exemplary mutant luciferases. TABLE III Protein SpectralProperties LucPplGR- peak shift width w.t. 545 0 72 V₂₁₄S * Q * Y * K *L * G * C * E * F * P * H * R * R₂₁₅H 562 17 82 Q 567 22 81 G 576 31 82T 576 31 84 M 582 37 83 P 588 43 91 S * Y * K * L * C * E * F * R₂₂₃L549 4 75 Q 549 4 73 R₂₂₃M 549 4 75 H 551 6 75 S * Y * K * G * C * E *F * P * V₂₂₄I 546 1 75 S 556 11 70 F 561 16 84 Y 565 20 87 L 578 33 94 H584 39 69 G 584 39 70 V₂₃₂E 554 9 83 V₂₃₆H 554 9 74 W 554 9 74 Y₂₃₇S 5538 73 C 554 9 74 L₂₃₈R 544 −1 72 M 555 10 75 Q 557 12 76 S 559 14 73 D568 23 76 H₂₄₂A 559 14 75 H₂₄₂S 561 16 74 F₂₄₄L 555 10 73 G₂₄₅S 558 1375 E 574 29 79 S₂₄₇H 564 19 72 Y 566 21 79 F 569 24 84 I₂₄₈R 544 −1 72 V546 1 72 F 548 3 74 T 554 9 75 S 558 13 80 N 577 32 90 H₃₄₈A 592 47 67 C593 48 66 N 597 52 67 Q 605 60 72 V₂₁₄C/V₂₂₄A 559 14 72 S₂₄₇F/F₂₄₆L 56722 79 S₂₄₇F/I₂₄₈C 586 41 84 S₂₄₇F/I₂₄₈T 596 51 80 T₂₃₃A/L₂₃₈M 555 10 75V₂₈₂I/I₂₈₃V 563 3 73 V₂₂₄F/R₂₁₅G 584 39 80 V₂₂₄F/R₂₁₅T 587 42 80V₂₂₄F/R₂₁₅V 589 44 80 V₂₂₄F/R₂₁₅P 597 52 81 V₂₂₄F/P₂₂₂S 564 3 86V₂₂₄F/Q₂₂₇E 583 38 85 V₂₂₄F/L₂₃₈V 575 30 85 V₂₂₄F/L₂₃₈M 576 31 87V₂₂₄F/S₂₄₇G 581 36 84 V₂₂₄F/S₂₄₇H 581 36 79 V₂₂₄F/S₂₄₇Y 595 50 88V₂₂₄F/S₂₄₇F 597 52 85

[0042] “Corresponding positions” in luciferases other than LucPplGR canbe determined either from alignments at the amino acid level that arealready known in the art (see, e.g., Wood et al., Science 244, 700-702(1989); Devine et al., Biochim. et Biophys. Acta 1173, 121-132(1993)) orby simply aligning at the amino acid level to maximize alignment ofidentical or conservatively substituted residues, and keeping in mind inparticular that amino acids 195-205 in the LucPplGR sequence are veryhighly conserved in all beetle luciferases and that there are no gapsfor more than 300 positions after that highly conserved 11-mer in anybeetle luciferase aminio acid sequence.

[0043] A “peroxisome-targeting-avoiding sequence at itscarboxy-terminus” means (1) the three carboxy-terminal amino acids ofthe corresponding wild-type luciferase are entirely missing from themutant; or (2) the three carboxy-terminal amino acids of thecorresponding wild-type luciferase are replaced with a sequence, of one,two or three amino acids that, in accordance with Sommer et al., supra,will reduce peroxisome-targeting by at least 50%. If the threecarboxy-terminal amino acids of the wild-type luciferase are replaced bya three-amino-acid peroxisome-targeting-avoiding sequence in the mutant,and if the sequence in the mutant is X₁X₂X₃, where X₃ iscarboxy-terminal, than X₁ is any of the twenty amino acids except A, C,G, H, N, P, Q, T and S, X₂ is any of the twenty amino acids except H, M,N, Q, R, S and K, and X₃ is any of the twenty amino acids except I, M, Yand L. Further, any one or two, or all three, of X₁, X₂, and X₃ could beabsent from the mutant (i.e., no amino acid corresponding to theposition). The most preferred peroxisome-targeting-avoiding sequence isIAV, where V is at the carboxy-terminus.

[0044] In another of its aspects, the invention entails a combination ofluciferases, in a cell (eukaryotic or prokaryotic), a solution (free orlinked as a reporter to an antibody, antibody-fragment, nucleic acidprobe, or the like), or adhererd to a solid surface, optionally throughan antibody, antibody fragment or nucleic acid, and exposed to asolution, provided that at least one of the luciferases is a mutant,both of the luciferases remain active in producing bioluminescence, andthe wavelengths of peak intensities of the bioluminescence of theluciferases differ because the amino acid sequences of the luciferasesdiffer at at least one of the positions corresponding to positions 214,215, 223, 224, 232, 236, 237, 238, 242, 244, 245, 247, 248, 282, 283 and348 in the amino acid sequence of LucPplGR, provided that one or both ofthe luciferases optionally have peroxisome-targeting-avoiding sequences.

[0045] In another of its aspects, the invention entails a DNA molecule,which may be an eukaryotic or prokaryotic expression vector, whichcomprises a segment which has a sequence which encodes a mutant beetleluciferase of the invention.

[0046] Most preferred among the DNAs of the invention are those withsegments which encode a preferred mutant luciferase of the invention.

[0047] From the description of the invention provided herein, theskilled will recognize many modifications and variations of what hasbeen described that are within the spirit of the invention. It isintended that such modifications and variations also be understood aspart of the invetion.

1 1 1632 base pairs nucleic acid double linear cDNA to mRNA1 no no 1 ATGATG AAG AGA GAG AAA AAT GTT GTA TAT GGA CCC GAA CCC CTA CAC 48 Met MetLys Arg Glu Lys Asn Val Val Tyr Gly Pro Glu Pro Leu His 5 10 15 CCC TTGGAA GAC TTA ACA GCA GGA GAA ATG CTC TTC AGG GCC CTT CGA 96 Pro Leu GluAsp Leu Thr Ala Gly Glu Met Leu Phe Arg Ala Leu Arg 20 25 30 AAA CAT TCTCAT TTA CCG CAG GCT TTA GTA GAT GTG TAT GGT GAA GAA 144 Lys His Ser HisLeu Pro Gln Ala Leu Val Asp Val Tyr Gly Glu Glu 35 40 45 TGG ATT TCA TATAAA GAG TTT TTT GAA ACT ACA TGC CTA CTA GCA CAA 192 Trp Ile Ser Tyr LysGlu Phe Phe Glu Thr Thr Cys Leu Leu Ala Gln 50 55 60 AGT CTT CAC AAT TGTGGA TAC AAG ATG AGT GAT GTA GTG TCG ATC TGC 240 Ser Leu His Asn Cys GlyTyr Lys Met Ser Asp Val Val Ser Ile Cys 65 70 75 80 GCG GAG AAC AAT AAAAGA TTT TTT GTT CCC ATT ATT GCA GCT TGG TAT 288 Ala Glu Asn Asn Lys ArgPhe Phe Val Pro Ile Ile Ala Ala Trp Tyr 85 90 95 ATT GGT ATG ATT GTA GCACCT GTT AAT GAG GGC TAC ATC CCA GAT GAA 336 Ile Gly Met Ile Val Ala ProVal Asn Glu Gly Tyr Ile Pro Asp Glu 100 105 110 CTC TGT AAG GTC ATG GGTATA TCG AGA CCA CAA CTA GTT TTT TGT ACA 384 Leu Cys Lys Val Met Gly IleSer Arg Pro Gln Leu Val Phe Cys Thr 115 120 125 AAG AAT ATT CTA AAT AAGGTA TTG GAG GTA CAG AGC AGA ACT GAT TTC 432 Lys Asn Ile Leu Asn Lys ValLeu Glu Val Gln Ser Arg Thr Asp Phe 130 135 140 ATA AAA AGG ATT ATC ATACTA GAT GCT GTA GAA AAC ATA CAC GGT TGT 480 Ile Lys Arg Ile Ile Ile LeuAsp Ala Val Glu Asn Ile His Gly Cys 145 150 155 160 GAA AGT CTT CCC AATTTT ATT TCT CGT TAT TCG GAT GGA AAT ATT GCC 528 Glu Ser Leu Pro Asn PheIle Ser Arg Tyr Ser Asp Gly Asn Ile Ala 165 170 175 AAC TTC AAA CCT TTACAT TAC GAT CCT GTT GAA CAA GTG GCA GCT ATC 576 Asn Phe Lys Pro Leu HisTyr Asp Pro Val Glu Gln Val Ala Ala Ile 180 185 190 TTA TGT TCG TCA GGCACA ACT GGA TTA CCG AAA GGT GTA ATG CAA ACT 624 Leu Cys Ser Ser Gly ThrThr Gly Leu Pro Lys Gly Val Met Gln Thr 195 200 205 CAT AGA AAT GTT TGTGTC CGA CTT ATA CAT GCT TTA GAC CCC AGG GTA 672 His Arg Asn Val Cys ValArg Leu Ile His Ala Leu Asp Pro Arg Val 210 215 220 GGA ACG CAA CTT ATTCCT GGT GTG ACA GTC TTA GTA TAT CTG CCT TTT 720 Gly Thr Gln Leu Ile ProGly Val Thr Val Leu Val Tyr Leu Pro Phe 225 230 235 240 TTC CAT GCT TTTGGG TTC TCT ATA AAC TTG GGA TAC TTC ATG GTG GGT 768 Phe His Ala Phe GlyPhe Ser Ile Asn Leu Gly Tyr Phe Met Val Gly 245 250 255 CTT CGT GTT ATCATG TTA AGA CGA TTT GAT CAA GAA GCA TTT CTA AAA 816 Leu Arg Val Ile MetLeu Arg Arg Phe Asp Gln Glu Ala Phe Leu Lys 260 265 270 GCT ATT CAG GATTAT GAA GTT CGA AGT GTA ATT AAC GTT CCA GCA ATA 864 Ala Ile Gln Asp TyrGlu Val Arg Ser Val Ile Asn Val Pro Ala Ile 275 280 285 ATA TTG TTC TTATCG AAA AGT CCT TTG GTT GAC AAA TAC GAT TTA TCA 912 Ile Leu Phe Leu SerLys Ser Pro Leu Val Asp Lys Tyr Asp Leu Ser 290 295 300 AGT TTA AGG GAATTG TGT TGC GGT GCG GCA CCA TTA GCA AAG GAA GTT 960 Ser Leu Arg Glu LeuCys Cys Gly Ala Ala Pro Leu Ala Lys Glu Val 305 310 315 320 GCT GAG ATTGCA GTA AAA CGA TTA AAC TTG CCA GGA ATT CGC TGT GGA 1008 Ala Glu Ile AlaVal Lys Arg Leu Asn Leu Pro Gly Ile Arg Cys Gly 325 330 335 TTT GGT TTGACA GAA TCT ACT TCA GCT AAT ATA CAC AGT CTT AGG GAT 1056 Phe Gly Leu ThrGlu Ser Thr Ser Ala Asn Ile His Ser Leu Arg Asp 340 345 350 GAA TTT AAATCA GGA TCA CTT GGA AGA GTT ACT CCT TTA ATG GCA GCT 1104 Glu Phe Lys SerGly Ser Leu Gly Arg Val Thr Pro Leu Met Ala Ala 355 360 365 AAA ATA GCAGAT AGG GAA ACT GGT AAA GCA TTG GGA CCA AAT CAA GTT 1152 Lys Ile Ala AspArg Glu Thr Gly Lys Ala Leu Gly Pro Asn Gln Val 370 375 380 GGT GAA TTATGC ATT AAA GGT CCC ATG GTA TCG AAA GGT TAC GTG AAC 1200 Gly Glu Leu CysIle Lys Gly Pro Met Val Ser Lys Gly Tyr Val Asn 385 390 395 400 AAT GTAGAA GCT ACC AAA GAA GCT ATT GAT GAT GAT GGT TGG CTT CAC 1248 Asn Val GluAla Thr Lys Glu Ala Ile Asp Asp Asp Gly Trp Leu His 405 410 415 TCT GGAGAC TTT GGA TAC TAT GAT GAG GAT GAG CAT TTC TAT GTG GTG 1296 Ser Gly AspPhe Gly Tyr Tyr Asp Glu Asp Glu His Phe Tyr Val Val 420 425 430 GAC CGTTAC AAG GAA TTG ATT AAA TAT AAG GGC TCT CAG GTA GCA CCT 1344 Asp Arg TyrLys Glu Leu Ile Lys Tyr Lys Gly Ser Gln Val Ala Pro 435 440 445 GCA GAACTA GAA GAG ATT TTA TTG AAA AAT CCA TGT ATC AGA GAT GTT 1392 Ala Glu LeuGlu Glu Ile Leu Leu Lys Asn Pro Cys Ile Arg Asp Val 450 455 460 GCT GTGGTT GGT ATT CCT GAT CTA GAA GCT GGA GAA CTG CCA TCT GCG 1440 Ala Val ValGly Ile Pro Asp Leu Glu Ala Gly Glu Leu Pro Ser Ala 465 470 475 480 TTTGTG GTT ATA CAG CCC GGA AAG GAG ATT ACA GCT AAA GAA GTT TAC 1488 Phe ValVal Ile Gln Pro Gly Lys Glu Ile Thr Ala Lys Glu Val Tyr 485 490 495 GATTAT CTT GCC GAG AGG GTC TCC CAT ACA AAG TAT TTG CGT GGA GGG 1536 Asp TyrLeu Ala Glu Arg Val Ser His Thr Lys Tyr Leu Arg Gly Gly 500 505 510 GTTCGA TTC GTT GAT AGC ATA CCA AGG AAT GTT ACA GGT AAA ATT ACA 1584 Val ArgPhe Val Asp Ser Ile Pro Arg Asn Val Thr Gly Lys Ile Thr 515 520 525 AGAAAG GAA CTT CTG AAG CAG TTG CTG GAG AAG AGT TCT AAA CTT TAA 1632 Arg LysGlu Leu Leu Lys Gln Leu Leu Glu Lys Ser Ser Lys Leu 530 535 540

We claim:
 1. An isolated DNA molecule comprising a segment having asequence which encodes a synthetic mutant beetle luciferase comprisingan amino acid sequence that differs from that of the correspondingwild-type luciferase by at least one amino acid substitution, theposition of the amino acid substitution corresponding to a position inthe amino acid sequence of LucPplGR of SEQ ID NO:2 selected from thegroup consisting of position 215, 224, 238, 242, 247 and 348, whereinthe mutant luciferase produces bioluminescence having a shift inwavelength of peak intensity of at least 1 nanometer relative to thebioluminescence produced by the wild-type luciferase, wherein for theencoded amino acid sequence, the corresponding wild-type luciferase isLucPplGR of SEQ ID NO:2, and wherein the encoded synthetic mutantluciferase includes an amino acid substitution selected from the groupconsisting of LucPplGR-R₂₁₅Q, -R₂₁₅S, -R₂₁₅Y, -R₂₁₅K, -R₂₁₅C, -R₂₁₅E,-R₂₁₅F, -H₂₄₂S, -H₃₄₈A, -V₂₂₄F/L₂₃₈M, and -V₂₂₄F/S₂₄₇Y.
 2. An isolatedDNA molecule according to claim 1, wherein the encoded mutant luciferasehas one amino acid substitution.
 3. An isolated DNA molecule accordingto claim 1, wherein the encoded mutant luciferase has two amino acidsubstitutions.
 4. An isolated DNA molecule according to claim 1, whereinthe encoded synthetic mutant luceriferase is LucPplGR-R₂₁₅Q.
 5. Anisolated DNA molecule according to claim 1, wherein encoded syntheticmutant luceriferase is LucPplGR-R₂₁₅S.
 6. An isolated DNA moleculeaccording to claim 1, wherein the encoded synthetic mutant luceriferaseis LucPplGR-R₂₁₅Y.
 7. An isolated DNA molecule according to claim 1,wherein the encoded synthetic mutant luceriferase is LucPplGR-R₂₁₅K. 8.An isolated DNA molecule according to claim 1, wherein the encodedsynthetic mutant luceriferase is LucPplGR-R₂₁₅C.
 9. An isolated DNAmolecule according to claim 1, wherein the encoded synthetic mutantluceriferase is LucPplGR-R₂₁₅E.
 10. An isolated DNA molecule accordingto claim 1, wherein the encoded synthetic mutant luceriferase isLucPplGR-R₂₁₅F.
 11. An isolated DNA molecule according to claim 1,wherein the encoded synthetic mutant luceriferase is LucPplGR-H₂₄₂S. 12.An isolated DNA molecule according to claim 1, wherein the encodedsynthetic mutant luceriferase is LucPplGR-H₃₄₈A.
 13. An isolated DNAmolecule according to claim 1, wherein the encoded synthetic mutantluceriferase is LucPplGR-V₂₂₄F/L₂₃₈M.
 14. An isolated DNA moleculeaccording to claim 1, wherein the encoded synthetic mutant luceriferaseis LucPplGR-V₂₂₄F/S₂₄₇Y.
 15. An isolated DNA molecule comprising asegment having a sequence which encodes a mutant beetle luciferasehaving an amino acid sequence that differs from that of thecorresponding wild-type luciferase LucPplGR by at least one amino acidsubstitution, wherein the encoded mutant luciferase is selected from thegroup consisting of LucPplGR-R₂₁₅Q, -R₂₁₅S, -R₂₁₅Y, -R₂₁₅K, -R₂₁₅C,-R₂₁₅E, -R₂₁₅F, -H₂₄₂S, -H₂₃₈A, -V₂₂₄F/L₂₃₈M, and -V₂₂₄F/S₂₄₇Y and theencoded mutant luciferase produces bioluminescence having a shift inwavelength of peak intensity of at least 1 nanometer relative to thebioluminescence produced by the wild-type luciferase.